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PubMed2006

Transglutaminase activity regulates osteoblast differentiation and matrix mineralization in MC3T3-E1 osteoblast cultures.

Transglutaminase (TG) enzymes and protein crosslinking have long been implicated in the formation of mineralized tissues. The aim of this study was to analyze the expression, activity and function of TGs in differentiating osteoblasts to gain further insight into the role of extracellular matrix protein crosslinking in bone formation. MC3T3-E1 (subclone 14) pre-osteoblast cultures were treated with ascorbic acid and beta-glycerophosphate to induce cell differentiation and matrix mineralization. Expression of TG isoforms was analyzed by RT-PCR. TG activity was assessed during osteoblast differentiation by in vitro biochemical assays and by in situ labeling of live cell cultures. We demonstrate that MC3T3-E1/C14 osteoblasts express two TG isoforms--TG2 and FXIIIA. Abundant TG activity was observed during cell differentiation which increased significantly after thrombin treatment, a result confirming the presence of FXIIIA in the cultures. Ascorbic acid treatment, which stimulated collagen secretion and assembly, also stimulated externalization of TG activity, likely from FXIIIA which was externalized upon this treatment as analyzed by immunofluoresence microscopy. Inhibition of TG activity in the cultures by cystamine resulted in complete abrogation of mineralization, attributable to decreased matrix accumulation and an arrested state of osteoblast differentiation as measured by decreased levels of bone sialoprotein, osteocalcin and alkaline phosphatase. Additional functional studies and substrate characterization showed that TG activity was required for the formation of a fibronectin-collagen network during the early stages of matrix formation and assembly. This network, in turn, appeared to be essential for further matrix production and progression of the osteoblast differentiation program, and ultimately for mineralization.

3T3 Cells↗
PubMed2006

Tissue transglutaminase crosslinks ataxin-1: possible role in SCA1 pathogenesis.

Transglutaminase type 2 (TG2) has recently been implicated in crosslinking of mutant huntingtin protein into aggregates. Here we show that TG2 also crosslinks spinocerebellar ataxia-1 (SCA1) gene product ataxin-1. HeLa cell lysates expressing GFP tagged ataxin-1 with 2, 30 or 82 glutamines showed covalent crosslinking of ataxin-1 when incubated with exogenously added TG2. This crosslinking was inhibited by TG2 inhibitor cystamine. SCA1 transgenic mice which overexpress the mutant ataxin-1 in cerebellar Purkinje cells showed elevated nuclear TG2 in the absence of ataxin-1 nuclear aggregates. The addition of purified TG2 to the nuclear extracts or addition of SCA1 nuclear TG2 to GFP-Q82 HeLa cell lysates resulted in the formation of insoluble aggregates. These data indicate that ataxin-1 is a substrate of TG2. Further, in SCA1 TG2 may translocate to the nucleus in response to nuclear accumulation of mutant ataxin-1 at early stages of the disease.

Animals↗
PubMed2003

Activation of in situ tissue transglutaminase by intracellular reactive oxygen species.

We have investigated the novel function of intracellular reactive oxygen species (ROS) in the activation of in situ tissue transglutaminase (tTGase) by lysophosphatidic acid (LPA) and transforming growth factor-beta (TGF-beta) in Swiss 3T3 fibroblasts. LPA induced a transient increase of intracellular ROS with a maximal increase at 10 min, which was blocked by ROS scavengers, N-acetyl-L-cysteine and catalase. LPA activated tTGase with a maximal increase at 1h, which was inhibited by cystamine and ROS scavengers. Incubation with exogenous H(2)O(2) activated tTGase. TGF-beta also activated tTGase with a maximal activation at 2h and the tTGase activation was inhibited by the ROS scavengers. Scrape-loading of C3 transferase inhibited the ROS production and in situ tTGase activation by LPA and TGF-beta, and the inhibitory effect of C3 transferase was reversed by exogenous H(2)O(2). Microinjection of GTPgammaS inhibited transamidating activity of tTGase stimulated by LPA, TGF-beta, and maitotoxin. These results suggested that intracellular ROS was essential for the activation of in situ tTGase in response to LPA and TGF-beta.

ADP Ribose Transferases↗
PubMed2000

Effects of nitric oxide donors on the afferent resting activity in the cephalopod statocyst.

The effects of bath applications of the nitric oxide (NO) donors sodium nitroprusside (SNP), diethylamine sodium (DEA), 3-morpholinosydnonimine (SIN-1), and S-nitroso-N-acetyl-penicillamine (SNAP) on the resting activity (RA) of afferent crista fibers were studied in isolated statocysts of the cuttlefish Sepia officinalis. The NO donors had three different effects: inhibition, excitation, and excitation followed by an inhibition. The SNAP analog N-acetyl-DL-penicillamine (xSNAP; with no NO moiety) had no effect. When the preparation was pre-treated with the NO synthase inhibitor N(G)-nitric-L-arginine methyl ester HCl (L-NAME), the NO donors were still effective. When the preparation was pre-treated with the guanylate cyclase inhibitors methylene blue (M-BLU) or cystamine (CYS), NO donors had only excitatory effects, whereas their effects were inhibitory only when pre-treatment was with the adenylate cyclase inhibitors nicotinic acid (NIC-A), 2',3'-dideoxyadenosine (DDA), or MDL-12330A. When pre-treatment was with a guanylate and an adenylate cyclase inhibitor combined, NO donors had no effect; in that situation, the RA of the afferent fibers remained and the preparation still responded to bath applications of GABA. Selective experiments with statocysts from the squid Sepioteuthis lessoniana and the octopod Octopus vulgaris gave comparable results. These data indicate that in cephalopod statocysts an inhibitory NO-cGMP and an excitatory NO-cAMP signal transduction pathway exist, that these two pathways are the key pathways for the action of NO, and that they have only modulatory effects on, and are not essential for the generation of, the RA.

Action Potentials↗
PubMed2000

The gamma-glutamylcysteine synthetase of Onchocerca volvulus.

The tripeptide glutathione (GSH) plays an important role in the maintenance of the intracellular thiol redox state and in detoxification processes. The intracellular GSH level depends on glutathione reductase as well as on GSH synthesis. The first and rate limiting step in the synthetic pathway is catalysed by gamma-glutamylcysteine synthetase (gamma-GCS). The gamma-GCS was partially purified from the filarial parasite Onchocerca volvoulus and preliminary steady state kinetics were performed. The Ki-value for L-buthionine-S,R-sulphoximine (BSO), a specific inhibitor of gamma-GCS, was determined to be 0.13 microM, which is 54-fold lower than the Ki-value for the mammalian enzyme. Filarial gamma-GCS was also inhibited by cystamine with a Ki-value of 3.9 microM compared with 22.2 microM determined for the rat enzyme. Further, the cDNA and the gene of the O. volvulus gamma-GCS were cloned and sequenced. The gene of 5762 bp is composed of 14 exons and 13 introns. Southern blot analysis indicates that the gamma-GCS gene is present as a single-copy gene. In accordance with Northern blot analysis, the entire cDNA sequence encompasses 2377 bp. At its 5' end a nematode-specific spliced leader 130 bp upstream of the first in frame methionine was identified. The cDNA encodes a polypeptide of 652 amino acids with 50 and 69% sequence identity to the human and the Caenorhabditis elegans counterparts, respectively. The filarial gamma-GCS is proposed as a potential drug target.

Amino Acid Sequence↗
PubMed2003

Identifying and modulating disulfide formation in the biopharmaceutical production of a recombinant protein vaccine candidate.

Structural conversion of the serotype A recombinant botulinum neurotoxin heavy chain fragment (rBoNTA(Hc)) produced intracellularly in Pichia pastoris yeast was observed and characterized during purification development efforts. A pH screening study captured the transformation stages of the original recovered species into its derived counterpart and a number of analytical tools such as peptide mapping by LC/MS confirmed the formation of a disulfide bond, especially in samples of neutral to basic pH. A cation exchange chromatographic method proved useful in following the incidence of the reaction in various rBoNTA(Hc) samples. The disulfide formation kinetics were characterized using a one-quarter quadratic factorial design, following the investigation and development of controlled oxidation conditions using cysteine and cystamine as the redox pair. Temperature, pH and concentration of the redox pair had a significant effect on the yield and rate of the disulfide formation. This controlled reaction was eventually introduced as a functional unit operation in the purification process. The summation of preliminary scale-up and potency data showed scalability and robustness in the production of an active disulfide-bonded form of a recombinant botulism vaccine candidate. The presence of the disulfide bond did not effect the vaccine potency and it enhanced the molecule's thermal stability.

Amino Acid Sequence↗
PubMedDate not supplied

Glucan as stimulator of hematopoiesis in normal and gamma-irradiated mice. A survey of the authors' results.

Glucan, a beta-1,3-linked polyglucose derived from the yeast Saccharomyces cerevisiae, is a broad spectrum enhancer of host defense mechanisms stimulating humoral and cell-mediated immunity. On the basis of these features, glucan has been tested by the authors' research group in experiments on gamma-irradiated mice. Two glucan forms, particulate and soluble, have been studied. Attention has been focused on various application regimens in relation to the time of irradiation (pre- or postirradiation application), the possibilities of using glucan in various radiation regimens (single or repeated irradiation), combined pharmacological therapy (joint administration of glucan with cystamine or inhibitors of prostaglandin synthesis), and on the negative side effects of therapy with glucan. Some studies included also experiments on unirradiated mice. The results have demonstrated the ability of glucan to influence positively the course of the acute radiation disease. Stimulation of hematopoiesis has been found to be the most important mechanism of glucan's radioprotective effects. In this communication, the results of 11 full-length articles are summarized and discussed.

Adjuvants, Immunologic↗
PubMed2000

Specific glutathione binding sites in pig cerebral cortical synaptic membranes.

Glutathione (gamma-glutamylcysteinylglycine) is a neuromodulator at glutamate receptors, but may also act as a neurotransmitter at sites of its own. The Na+-independent binding of [3H]glutathione to pig cortical synaptic membranes was characterized here using glycine, cysteine analogs, dipeptides and glutathione derivatives, and ligands selective for known glutamate receptors. L-Glutamate, pyroglutamate, quinolinate, (S)-5-fluorowillardiine and 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione were weak inhibitors at concentrations of 0.5 or 1 mM. D-Glutamate, L- and D-aspartate, glutamine, quisqualate, kynurenate, other N-methyl-D-aspartate receptor ligands and non-N-methyl-D-aspartate receptor ligands failed to displace [3H]glutathione. Except for weak inhibition by D-serine (0.5 mM), glycine and other ligands of the glycine co-activatory site in the N-methyl-D-aspartate receptors had no displacing effect. Similarly, metabotropic glutamate group I, II and III receptor agonists and antagonists and compounds acting at the glutamate uptake sites were generally inactive. Glutathione, oxidized glutathione, S-nitrosoglutathione, gamma-L-glutamylcysteine, cysteinylglycine, cysteine, cysteamine and cystamine were the most potent displacers (IC50 values in the micromolar range), followed by dithiothreitol, glutathione sulfonate and the S-alkyl derivatives of glutathione (S-methyl-, -ethyl-, -propyl-, -butyl- and -pentylglutathione). L-Homocysteinate and aminomethanesulfonate exhibited a moderate efficacy. Thiokynurenate, a cysteine analog and an antagonist at the N-methyl-D-aspartate receptor glycine co-activatory site, was a potent activator of glutathione binding. At 1 mM, some dipeptides also slightly activated the binding, gamma-L-glutamylleucine and gamma-L-glutamyl-GABA being the most effective. The specific binding sites for glutathione in brain synaptic membranes are not identical to any known excitatory amino acid receptor. The cysteinyl moiety is crucial in the binding of glutathione. The oxidation or alkylation of the cysteine thiol group reduces the binding affinity. The strong activation by thiokynurenate may indicate that the glutathione receptor protein contains a modulatory site to which co-agonists may bind and allosterically activate glutathione binding. The novel population of specific binding sites of glutathione gives rise to the possibility that they may have profound effects on synaptic functions in the mammalian central nervous system. The glutathione binding sites may be an important, and for the most part unrecognized, component in signal transduction in the brain.

Animals↗
PubMed1997

Glutathione-dependent factors and inhibition of rat liver microsomal lipid peroxidation.

The effects of reduced glutathione (GSH) and glutathione disulfide (GSSG) on lipid peroxidation were investigated in rat liver microsomes containing deficient or adequate amounts of alpha-tocopherol (alpha-TH). Rates of formation of thiobarbituric acid reactive substances (TBARS) as well as rates of consumption of alpha-TH and O2 were decreased by GSH and were more pronounced in the NADPH-dependent assay system than in the ascorbate-dependent system. The GSH-dependent inhibition of lipid peroxidation was potentiated by GSSG in the NADPH-dependent assay system, but it had no effect in the nonenzymatic system. Diphenyliodonium chloride, an inhibitor of NADPH cytochrome P-450 reductase, completely prevented lipid peroxidation in the NADPH-dependent assay system whereas it had no effect on the ascorbate-dependent system. This is further evidenced by the fact that purified rat liver microsomal NADPH cytochrome P-450 reductase (EC 1.6.2.4) was inhibited approximately 24% and 52% by 5 mM GSH and 5 mM GSH + 2.5 mM GSSG, respectively. Glutathione disulfide alone had no effect on reductase activity. Similarly, other disulfides such as cystine, cystamine and lipoic acid were without effect on reductase activity. These results clearly delineate different mechanisms underlying the combined effects of GSH and GSSG on microsomal lipid peroxidation in rat liver. One mechanism involves recycling of microsomal alpha-TH by GSH during oxidative stress via a labile protein, ostensibly associated with "free radical reductase" activity. A second glutathione-dependent mechanism appears to be mediated through the inhibition of NADPH cytochrome P-450 reductase. The enhanced inhibition by GSH + GSSG of microsomal lipid peroxidation in the NADPH-dependent assay system suggests suppression of the initiation phase at the level of NADPH cytochrome P-450 reductase which is independent of microsomal alpha-TH.

Animals↗
PubMed1998

Modification of enzyme sulfhydryl groups suppresses UV-induced mutagenesis depending on the nucleotide excision repair system in Escherichia coli B/r WP2.

S-Methyl methanethiosulfonate (MMTS), which was isolated from cauliflower (Brassica oleracea var. botrytis) homogenate as a potent bio-antimutagen, has been used as an enzyme-sulfhydryl (SH) temporary blocking agent in modification studies of enzyme activities. We examined whether 23 kinds of MMTS-related compounds have a suppressing effect on UV mutagenesis in Escherichia coli B/r WP2. Disulfide derivatives of diphenyl, 2.2'-dipyridine and 4.4'-dipyridine, and N-ethyl maleimide (NEM), which temporarily or tightly block sulfhydryl (SH)-groups, showed similar suppressing effect in E. coli B/r WP2, but not in WP2s hcr- (uvrA-) in the range of nanomolar/plate as MMTS previously did. Cystamine sulfate, methyl methylsulfinylmethyl sulfide and S-methyl-L-cysteinesulfoxide moderately suppressed, and diallyl disulfide and glutathione (oxidized form) weakly suppressed UV mutagenesis in E. coli B/r WP2 in the range of micromolar/plate. MMTS and phorone, a glutathione (GSH)-depleting agent, lowered the intracellular GSH level in E. coli B/r WP2, but phorone did not inhibit UV-induced mutation. These results indicate that the target for SH-modification is enzyme-SHs but not GSH, and that the direct or indirect modification of enzyme-activity by SH-blocking might be involved in the antimutagenesis through a pathway associated with the DNA-excision repair system.

Antimutagenic Agents↗
PubMed2001

Investigation of highly sensitive piezoelectric immunosensors for 2,4-dichlorophenoxyacetic acid.

The improved highly sensitive piezoelectric immunosensor has been developed and evaluated using a model interaction of antibody with the model hapten-herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). For immobilization of 2,4-D, the self-assembled layers of cystamine, 4-aminothiophenol or 3,3'-dithio-bis(propionic acid N-hydroxysuccinimide ester) were formed on smooth and rough crystals coated with gold or silver electrodes. The immunochemical interactions performed well in all cases, the aminothiophenol on gold was chosen as the optimum with regard to regeneration of immunosensing surfaces. The kinetics of interaction of surface-bound 2,4-D with free antibody provided significantly higher kinetic parameters (kinetic association rate constant) when using optically smooth crystals compared to common rough crystal. Therefore, the smooth crystal should be preferred for future kinetic studies. The competitive assay of the herbicide 2,4-D achieved a limit of detection of 10 ng/l using the monoclonal anti-2,4-D antibody F6C10. Finally, a direct assay format has been evaluated using a thicker layer of glutaraldehyde-crosslinked antibody on the sensing surface. The direct binding of a small herbicide molecule was followed in real time. The detected concentration of 2,4-D (5 microg/l) was low enough for future direct monitoring of this herbicide in water.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2003

Flow injection amperometric detection of OP nerve agents based on an organophosphorus-hydrolase biosensor detector.

A flow-injection system with an organophosphorus-hydrolase (OPH)-biosensor detector has been developed and characterized for the rapid detection of organophosphorus (OP) nerve agents. The enzyme was immobilized onto a thin-film gold detector through a cystamine-glutaraldehyde coupling. Factors influencing the performance were optimized. The resulting flow system offered a fast, sensitive, selective, and stable response. The peak current increased linearly with the concentration of paraoxon and methyl parathion over the 1-10 microM range (sensitivity, 2.29 and 1.04 nA/microM, respectively). The OPH-biosensor flow injection systems offered low detection limits (e.g. 0.1 microM paraoxon), along with a good precision (R.S.D. of 3.6% for 20 successive injections of a 1.0 microM paraoxon solution). The OPH-biosensor flow detector offers great promise for rapid field screening of OP pesticides and nerve agents.

Aryldialkylphosphatase↗
PubMed1998

Fully integrated biocatalytic electrodes based on bioaffinity interactions.

Integrated bioelectrocatalytically active electrodes are assembled by the deposition of enzymes onto respective electrically contacted affinity matrices and further cross-linking of the enzyme monolayers. A catalyst-NAD(+)-dyad for the binding of the NAD(+)-dependent enzymes and cytochrome-like molecules for the binding of the heme-protein-dependent enzymes are used to construct integrated electrically contacted biocatalytic systems. NAD(+)-dependent lactate dehydrogenase (LDH) is assembled onto a pyrroloquinoline quinone-NAD+ monolayer. The redox-active monolayer is organized via covalent attachment of pyrroloquinoline quinone (PQQ) to a cystamine monolayer associated with a Au-electrode, followed by covalent linkage of N6-(2-aminoethyl)-NAD+ to the monolayer. The interface modified with the PQQ-NAD(+)-dyad provides temporary affinity binding for LDH and allows cross-linking of the enzyme monolayer. The cross-linked LDH is bioelectrocatalytically active towards oxidation of lactate. The bioelectrocatalyzed process involves the PQQ-mediated oxidation of the immobilized NADH. Integrated, electrically contacted bioelectrodes are produced by the affinity binding and further cross-linking of nitrate reductase (NR) (cytochrome-dependent, E.C. 1.9.6.1 from E. coli) or CoII-protoporphyrin IX reconstituted myoglobin (CoII-Mb) atop the microperoxidase-11 (MP-11) monolayer associated with a Au-electrode. The MP-11 monolayer provides an affinity interface for the temporary binding of the enzymes, that allows the cross-linkage of the enzyme molecules. The MP-11 assembly acts as electron transfer mediator for the reduction of the secondary enzyme layer. The integrated bioelectrodes consisting of NR and CoII-Mb show catalytic activities for NO3- reduction and acetylene-dicarboxylic acid hydrogenation, respectively. Two FeIII-protoporphyrin IX units are reconstituted into a four alpha-helix bundle de novo protein assembled as a monolayer on a Au-electrode. Vectorial electron transfer proceeds in the synthetic heme-protein monolayer. Cross-linking of an affinity complex generated between the FeIII-protoporphyrin IX reconstituted de novo protein monolayer and NR yields an integrated, electrically contacted enzyme electrode that stimulates the bioelectrocatalyzed reduction of nitrate.

Animals↗
PubMed1998

Search for alpha 1-adrenoceptor subtypes selective antagonists: design, synthesis and biological activity of cystazosin, an alpha 1D-adrenoceptor antagonist.

Two novel quinazolines (2 and 3) related to both prazosin and its open analogue 1 were synthesized, and their biological profile at alpha 1-adrenoceptor subtypes was assessed by functional assays in rat isolated tissues, namely prostatic vas deferens (alpha 1A), spleen (alpha 1B) and aorta (alpha 1D). Furthermore, the binding profile of 3 was assessed at native alpha 2 and D2 receptors, and cloned human 5-HT1A receptors, in comparison to prazosin, (+)-cyclazosin, 1 and BMY 7383. It turned out that the cystamine-bearing quinazoline 3 (cystazosin) has a reversed affinity profile relative to (+)-cyclazosin owing to a higher affinity for alpha 1D-adrenoceptors and a significantly lower affinity for the alpha 1A and alpha 1B subtypes. Furthermore, in comparison to BMY 7378, cystazosin (3) displays a much better specificity profile since it has lower affinity for D2 and 5-HT1A receptors.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed2000

A novel biotinylated suicide inhibitor for directed molecular evolution of lipolytic enzymes.

A bifunctional activity label (8) for directed molecular evolution of lipolytic enzymes has been designed and synthesized. The structure is composed of a 4-nitrophenyl activated phosphonate, that is, a suicide substrate of lipases/esterases, connected to a biotin moiety through a spacer containing a disulfide bridge. The phosphonate (3) was prepared by Michaelis-Arbuzov reaction of trimethylsilyl-protected 11-bromoundecanol (2) with triethyl phosphite. The deprotected omega-hydroxyalkylphosphonate (4) was transformed into an active N-hydroxysuccinimide carbonate (5) followed by 4-nitrophenyl activation of the phosphonate using standard procedures. The biotinylated phosphonate inhibitor (8) was then synthesised by coupling the phosphonate inhibitor (6) to the epsilon-amino-caproic acid and cystamine containing biotinyl spacer (7). The function of all relevant groups of the final activity label (8) (biotin-label, cleavable disulfide bridge, phosphonate-inhibitor) have been successfully tested with the commercial lipase Lipolase (Novo Nordisk). Hence, a tool for directed molecular evolution of lipolytic enzymes has been developed.

Affinity Labels↗
PubMed1997

Piezoelectric immunosensors for urine specimens of Chlamydia trachomatis employing quartz crystal microbalance microgravimetric analyses.

The assembly of a biosensor for Chlamydia trachomatis based on the microgravimetric quartz crystal microbalance (QCM) analysis of the bacteria association to an antibody-functionalized electrode is described. The sensing interfaces consist of a primary cystamine monolayer assembled onto Au electrodes associated with the quartz crystal. The monolayer is further modified with sulfosuccinylimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB) and the goat IgG-anti-mouse IgG Fc-specific Ab or the fragmented F(ab')2 anti-mouse IgG Ab that act as sublayers for the association of the sensor-active anti-C. trachomatis LPS-Ab. Bacteria in the concentration range from 260 ng.mL-1 to 7.8 micrograms.mL-1 are sensed by the functionalized crystals. The association of C. trachomatis to the sensing interface can be confirmed and amplified via interaction of the crystal with various anti-C. trachomatis antibodies. Urine-pretreated functionalized quartz crystals are applied in the analysis of C. trachomatis in urine samples. The sensitivity limits of the electrodes for sensing the bacteria in urine samples corresponds to approximately 260 ng.mL-1. The functionalized crystals assembled via association of anti-C. trachomatis LPS-Ab to the fragmented F(ab')2 anti-mouse IgG Ab reveal long-term stability upon storage at 4 degrees C.

Antibodies, Bacterial↗
PubMed2000

Redox hydrogel-based amperometric bienzyme electrodes for fish freshness monitoring

This work presents the design and optimization of amperometric biosensors for the determination of biogenic amines (e.g., histamine, putrescine, cadaverine, tyramine, cystamine, agmatine, spermidine), commonly present in food products, and their application for monitoring of freshness in fish samples. The biosensors were used as the working electrodes of a three-electrode electrochemical cell of wall-jet type, operated at -50 mV vs Ag/AgCl, in a flow injection system. Two different bienzyme electrode designs were considered, one based on the two enzymes [a newly isolated and purified amine oxidase (AO) and horseradish peroxidase (HRP)] simply adsorbed onto graphite electrodes, and one when they were cross-linked to an Os-based redox polymer. The redox hydrogel-based biosensors showed better biosensors characteristics, i.e., sensitivity of 0.194 A M-1 cm-2 for putrescine and 0.073 A M-1 cm-2 for histamine, and detection limits (calculated as three times the signal-to-noise ratio) of 0.17 microM for putrescine and 0.33 microM for histamine. The optimized redox hydrogel-based biosensors were evaluated in terms of stability and selectivity, and were used for the determination of total amine content in fish samples kept for 10 days in different conditions.

Journal Article↗
PubMedDate not supplied

Synthesis of a stable and specific surface plasmon resonance biosensor surface employing covalently immobilized peptide nucleic acids.

Biosensors allow the real-time and label-free observation of biochemical reactions between various ligands including antigen-antibody reactions and nucleic acids hybridizations. In our studies, we used a surface plasmon resonance biosensor to elucidate the hybridization characteristics of a peptide nucleic acid (PNA) ligand immobilized on sensor surfaces either through covalent or streptavidin-biotin coupling. A biotin-labeled PNA was employed in the latter approach whereas the covalent immobilization included the following steps: A maleimide group was attached to the N-terminal of the PNA using N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). To generate free thiol groups for coupling, a carboxylated dextran matrix of the sensor surface was activated with N-hydroxysuccinimide (NHS) and N-ethyl-N'-(dimethylaminopropyl)-carbodiimide (EDC) and thiolated by addition of cystamine dihydrochloride followed by reduction with 1, 4-dithioerythrite (DTE). Finally, the modified PNA was coupled to the sulfhydryl groups of the activated dextran matrix. Repetitive hybridizations of a single-stranded synthetic DNA oligomer to the PNAs demonstrated the superior stability of covalent immobilization compared to noncovalent immobilization. Differentiation of point mutations in the analyte molecule was accomplished at 40 degrees C using guanidine thiocyanate concentrations of 1.5-1.7 M. In further experiments, we showed that a perfectly matched PNA allows the detection of a single-stranded DNA at a sensitivity of less than 1% in a background of single-stranded DNA having a single C to T point mutation in the region complementary to the PNA. Consequently, covalently bound PNAs provide a stable and reproducible environment for the development of mutation-specific DNA analysis assays.

Base Sequence↗